Project Summary Lyme disease is a progressive disease that, in later stages, can lead to debilitating permanent damage to the nervous and musculoskeletal systems. Early treatment with an appropriate course of antibiotics is highly effective at clearing infection and preventing disease progression. Therefore, early diagnosis is critical for ensuring patient outcome. However, most current IgM and IgG serological assays are not sensitive enough to detect disease at the time most patients seek medical attention. More effective alternatives are necessary. A T cell based cytokine release assay offers a unique and innovative alternative to serological assays. Unlike antibody responses, which may remain elevated for years after pathogen clearance, the T cell response rapidly wanes, with the cessation of effector function and a collapse in T cell numbers. Thus, monitoring the T cell activity may provide a more sensitive measure of active disease and pathogen clearance than serum antibody responses. In phase I, we identified unique peptides derived from several antigens expressed by Borrelia burgdorferi during the course of human disease. Using a mixture of peptides from four of these antigens, we generated a prototype assay that induced detectable secretion of IFN? following overnight stimulation of whole blood obtained from patients with well-characterized early Lyme disease, and observed that IFN? secretion was significantly decreased in the same patients 60 days after antibiotic therapy, indicating successful clearance of the bacteria. These data demonstrate concrete proof of principle that a cytokine release assay for the diagnosis of early Lyme disease can be created and can be successful. In this phase II application we will optimize this assay by 1) selecting additional peptide-epitopes that stimulate release of IFN? from T cells activated in response to infection with Borrelia spp, 2) confirming specificity by evaluating samples from patients with potential cross-reactive illnesses, and 3) evaluating the performance of a QuantiFERON-like test before and after treatment of all stages of Lyme disease. This will result in the creation of a finalized cytokine release assay for Lyme disease for FDA submission.